Figure 1: Ox-LDL induces VSMC-derived foam cell death, causes mtROS
accumulation, reduces ΔΨm, and upregulates pro-atherosclerosis factor
expression. VSMCs were treated for 72 h with 5 µg/mL
cyclodextrin-cholesterol complex to establish VSMC-derived foam cells,
which were subsequently treated with different concentrations of ox-LDL
for 24 h. (a) Viability of VSMC-derived foam cells was measured using
the CCK8 assay. (b) LDH release was evaluated using a cytotoxicity
detection kit. (c) Mitochondrial ROS levels were detected using the
MitoSOX™ Red mitochondrial superoxide indicator after treating
VSMC-derived foam cells for 6 h with different concentrations of ox-LDL.
(d) A JC-1 staining kit was used to measure mitochondrial membrane
potential after treating VSMC-derived foam cells for 6 h with different
concentrations ox-LDL. (e) Real-time PCR analysis was performed to
measure the mRNA levels of atherosclerosis-related factors, namely,
MYH11, CD36, CD47, ICAM-1, VCAM-1, IL-1β, IL-18, MCP-1, MMP9, and PCSK9.
Data are presented as mean ± SD of three independent experiments. *p
< 0.05 vs. the control group; **p < 0.01 vs. the
control group.
3.2. Ox-LDL (10 μg/mL) Induces VSMC-Derived Macrophage-Like Foam
Cell Necroptosis
Local inflammation in plaques plays an important role in atheroma
progression. Because apoptotic and necroptotic cell death have distinct
effects on inflammation, the type of cell death induced by ox-LDL needs
to be determined. To illustrate the pattern of ox-LDL-induced cell
death, cell morphology in each treatment group was examined using
electron microscopy. The appearance of plasma membrane destruction and
organelle fragmentation or swelling in 10 μg/mL ox-LDL-treated
VSMC-derived macrophage-like foam cells revealed that the cells were
necrotic (Fig. 2a). We used Annexin V and PI staining to determine the
cell death rate (Fig. 2b). VSMC-derived macrophage-like foam cells
without any treatment exhibited cell death, and PI-positive cells
accounted for a major portion of these cells. As PI uptake is an
indicator of the loss of cell membrane integrity, according to the
feature of necroptosis cells, we calculated PI-positive cell rate and
found that 10 μg/ mL ox-LDL treatment significantly promoted
VSMC-derived macrophage-like foam cell necroptosis (Fig. 2c).